This blog is meant to allow Fragment-based Drug Design Practitioners to get together and discuss NON-CONFIDENTIAL issues regarding fragments.

17 June 2015

Fragments vs HIV Reverse Transcriptase - again

Some targets are so heavily studied that you would think
there is nothing left to discover. HIV-1 Reverse Transcriptase (HIV-1 RT) is
one of these, with 13 marketed drugs against it: half of all anti-HIV drugs.
But as Gilda Tachedjian and collaborators at Burnet Institute, Monash
University, the University of Pittsburgh, and the University of Melbourne show
in a recent (and open-access) paper in Proc.
Nat. Acad. USA, there are still new insights to be learned about this
target.

The researchers started with an STD NMR screen of 630
Maybridge fragments, each at ~350 µM in pools of up to five. This gave 84 hits
– a healthy 13% hit rate. However, when these were tested in a functional assay
(RNA-dependent DNA polymerase activity, or RDDP) only 12 showed significant
inhibition, of which 6 were better than 1 mM. Testing 14 related compounds led
to 2 more hits, for a total of 8 fragments with IC50s from ~70-750
µM. However, one showed signs of aggregation in dynamic light scattering and
was not further pursued.

Since HIV-1 RT has been the object of such intensive
research, the team looked at the similarity of their fragments to known
binders, including those from previous fragment screening. Surprisingly, their
hits turned out to be quite distinct.

Next, the researchers looked at the effect of their
fragments on the DNA-dependent DNA
polymerase activity of HIV-1 RT, and happily found results similar to the RDDP assay above. The 5
most potent fragments were also tested against three clinically important
mutants of HIV-1 RT, and while two of them showed reduced activity, the other
three were either as potent or even more so. Testing these against unrelated
polymerases revealed that they are not merely promiscuous inhibitors.

Of course, functional activity at high concentrations can
have all sorts of causes, so the researchers performed a battery of careful
enzyme kinetics experiments to ascertain the mechanisms. One fragment turned
out to be competitive with respect to deoxynucleotide triphosphate substrate,
even though it looks nothing like a nucleotide. Another is competitive with the
DNA substrate. In other words, both these fragments operate through different mechanisms
of action from clinically approved HIV-1 RT inhibitors.

One of the most potent fragments is a p-hydroxyaniline, which the researchers recognized as a PAINS compound (it can form reactive quinones). However, freshly prepared samples of
this fragment were just as active as samples that had been stored in DMSO for
months. Also, an analog without the ability to form a quinone was still active,
albeit less so.

The p-hydroxyaniline
fragment also showed activity in a cell-based assay. Just as with biochemical
assays, cell-based assays are also susceptible to false positives, but the kinetics
of viral inhibition were consistent with inhibition of HIV-1 RT rather than
other other mechanisms. Further work on the compound may be merited; these are
exactly the kinds of investigations necessary to decide if an interesting PAINS
molecule is worth pursuing.

Unfortunately there is no crystallographic or detailed NMR
structural information as to how these molecules actually bind. Previous work
has identified multiple fragment binding sites on HIV-1 RT, so further work should
eventually reveal how these molecules interact with the protein.

In the end this paper shows that, even in the absence
of structure, it is possible to learn a great deal about how fragments inhibit
an enzyme. It is also a useful reminder that fragment-based approaches can
identify new types of inhibitors even for a target that has been intensively –
and successfully – studied for decades.